A scheme is described that should allow us to select mutations in synaptic transmission. The shibire mutation of Drosophila melanogaster is a temperature-sensitive paralytic mutant that causes nerve terminals to be depleted of synaptic vesicles. When flies are held at a temperature just below their paralytic temperature, a small reduction in the efficiency of transmitter release will cause paralysis. This screen has been used to isolate 93 mutants, enhancer of shibire or E(shi), that display this phenotype when heterozygous. We wish to screen these mutants biochemically to identify those that affect synaptic vesicle biogenesis or function. Synaptic vesicles, isolated from adult Drosophila brain, will be identified by their homogeneous size, their content of synaptic vesicle marker proteins and their absence from shibire flies held at non-permissive temperatures. The ability of our synaptic vesicle assay to recognize defects in endocytosis will be tested using alleles of shibire with different temperature thresholds for paralysis. The enhancer mutations will then be screened to identify mutations that affect recycling of synaptic vesicles from the plasma membrane, or the probability of synaptic vesicle release. If the rationale is correct, selection of shibire enhancers will allow us for the first time to select mutations in regulated secretion.